Thrombin-Binding Antibody Molecules And Uses Thereof

ABSTRACT

This invention relates to isolated antibodies which recognize the exosite 1 epitope of thrombin and selectively inhibit thrombin without promoting bleeding. These antibody molecules may be useful in the treatment and prevention of thrombosis, embolism and other conditions mediated by thrombin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/363,396, filed Mar. 25, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/976,512, filed May 10, 2018, which is acontinuation of U.S. patent application Ser. No. 15/348,250, filed Nov.10, 2016, now U.S. Pat. No. 9,988,463, which is a divisional of U.S.patent application Ser. No. 14/363,514, filed Jun. 6, 2014, now U.S.Pat. No. 9,518,129, which is an application filed under Section 371 ofInternational Patent Application No. PCT/GB2012/053140, filed Dec. 14,2012, which claims benefit of priority to GB 1121513.4, filed Dec. 14,2011. The contents of these applications are hereby incorporated byreference in their entireties.

REFERENCE TO A SEQUENCE LISTING

This instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 7, 2021, isnamed 103693_002546_SL.txt and is 8979 bytes in size.

TECHNICAL FIELD

This invention relates to antibody molecules that inhibit thrombin.

BACKGROUND

Blood coagulation is a key process in the prevention of bleeding fromdamaged blood vessels (haemostasis). However, a blood clot thatobstructs the flow of blood through a vessel (thrombosis) or breaks awayto lodge in a vessel elsewhere in the body (thromboembolism) can be aserious health threat.

A number of anticoagulant therapies are available to treat pathologicalblood coagulation. A common drawback of these therapies is an increasedrisk of bleeding (Mackman (2008) Nature 451(7181): 914-918). Manyanticoagulant agents have a narrow therapeutic window between the dosethat prevents thrombosis and the dose that induces bleeding. This windowis often further restricted by variations in the response in individualpatients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the binding and elution of the IgA on humanthrombin-Sepharose® column. FIG. 1A shows an elution profile for IgA(narrow peak) from a thrombin-Sepharose® column using a pH gradient(neutral to low, indicated by upward sloping line). FIG. 1B shows anative blue gel showing total IgA load, flow-through from the humanthrombin column and eluate following elution at low pH.

FIG. 2 shows a non-reducing SDS-PAGE gel which indicates that the IgAbinds thrombin but not prothrombin. In this pull-down assay, lectinagarose is used to bind to IgA in the presence of thrombin orprothrombin. The supernatant is then run on an SDS gel. Lane 1 is sizestandards; lane 2 shows a depletion of thrombin from the supernatant;Lane 3 shows that depletion is dependent on the presence of the IgA;Lanes 3 and 4 show that prothrombin is not depleted, and therefore doesnot bind to the IgA.

FIG. 3 shows the relative rate of 52238 cleavage by thrombin in thepresence or absence of IgA (i.e. a single slope of Abs405 with time forS2238 hydrolysis). This indicates that the IgA does not bind at thethrombin active site.

FIG. 4 shows the results of binding studies which indicate that the IgAcompetes with the fluorescently labelled dodecapeptide hirugen forbinding to thrombin.

FIG. 5 shows the effect of the IgA on the cleavage of S2238 by thrombin.This analysis allows the estimate of Kd for the IgA-thrombin interactionof 12 nM.

FIG. 6 shows an SDS-PAGE gel of whole IgA and Fab fragments underreducing and non-reducing (ox) conditions. The non-reduced IgA is shownto have a molecular weight of between 100-200 kDa and the non-reducedFab has a molecular weight of about 50 kDa.

FIG. 7 shows the crystal structure of Thrombin-Fab complex showinginteraction between the exosite 1 of thrombin and HCDR3 of the Fabfragment.

FIG. 8 shows detail of crystal structure showing interaction betweenspecific residues of thrombin exosite 1 and HCDR3 of the Fab fragment.

FIG. 9 shows fluorescence microscopy images of FeCl₃ induced blood clotsin femoral vein injuries in C57BL/6 mice injected with FITC labelledfibrinogen taken at between 2 and 30 minutes. 100 μl of PBS wasadministered (vehicle control).

FIG. 10 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice injected with FITClabelled fibrinogen and 40 nM (final concentration in mouse blood,equivalent to a dose of approximately 0.6 mg/Kg) anti-exosite 1 IgA (100μl in PBS).

FIG. 11 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice injected with FITClabelled fibrinogen and 80 nM (final concentration in mouse blood,equivalent to a dose of approximately 1.2 mg/Kg) anti-exosite 1 IgA (100μl in PBS), and a region outside of injury site for comparison.

FIG. 12 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice injected with FITClabelled fibrinogen and 200 nM (final concentration in mouse blood,equivalent to a dose of approximately 3 mg/Kg) anti-exosite 1 IgA (100μl in PBS), and a region outside of injury site for comparison.

FIG. 13 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice injected with FITClabelled fibrinogen and 400 nM (final concentration in mouse blood,equivalent to a dose of approximately 6 mg/Kg) anti-exosite 1 IgA (100μl in PBS).

FIG. 14 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice treated with FITClabelled fibrinogen and 4 μM (final concentration in mouse blood,equivalent to a dose of approximately 60 mg/Kg) anti-exosite 1 IgA (100μl in PBS).

FIG. 15 shows a quantitation of the dose response to anti-exosite 1 IgAfrom the fluorescent images shown in FIGS. 9 to 13.

FIG. 16 shows tail bleed times in control C57BL/6 mice and in micetreated with increasing amounts of anti-exosite 1 IgA. The secondaverage excludes the outlier.

FIG. 17 shows the results of tail clip assays on wild-type male C57BL/6mice (n=5) after injection into tail vein with either IgA or PBS. 15 minafter injection, tails were cut at diameter of 3 mm and blood lossmonitored over 10 min.

FIG. 18A to 18D show the results of an FeCl₃ carotid artery occlusionmodel on 9 week old WT C57BL/6 male mice injected as previously with 400nM anti-thrombin IgA (final concentration in blood, equivalent to a doseof approximately 6 mg/Kg) or PBS 15 min prior to injury with 5% FeCl₃for 2 min. FIG. 18A shows results for a typical PBS-injected mice(occlusion in 20 min) and FIGS. 18B, 18C and 18D show examples ofresults for mice treated with 400 nM anti-thrombin IgA (no occlusion).

FIG. 19 shows thrombin times (i.e. clotting of pooled plasma) withincreasing concentrations of IgG and IgA of the invention, upon additionof 20 nM human thrombin.

FIG. 20 shows the binding of synthetic IgG to immobilized thrombin (onForteBio® Octet Red instrument).

FIG. 21 shows a typical Octet trace for the binding of 24 nM S195Athrombin to immobilized IgG showing the on phase, followed by an offphase. The black line is the fit.

FIG. 22 shows an Octet trace of 500 nM prothrombin with a tip loadedwith immobilized IgG. The same conditions were used as the experimentwith thrombin in FIG. 21. There is no evidence of binding, even at thishigh concentration.

SUMMARY

The present invention relates to the unexpected finding that antibodymolecules which recognize the exosite 1 epitope of thrombin selectivelyinhibit thrombin without promoting bleeding. These antibody moleculesmay be useful in the treatment and prevention of thrombosis, embolismand other conditions mediated by thrombin.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

An aspect of the invention provides an isolated antibody molecule thatspecifically binds to exosite 1 of thrombin.

Isolated anti-exosite 1 antibody molecules may inhibit thrombin in vivowithout promoting or substantially promoting bleeding or haemorrhage,i.e. the antibody molecules do not inhibit or substantially inhibitnormal physiological responses to vascular injury (i.e. haemostasis).For example, haemostasis may not be inhibited or may be minimallyinhibited by the antibody molecules (i.e. inhibited to an insignificantextent which does not affect the well-being of patient or requirefurther intervention). Bleeding may not be increased or may be minimallyincreased by the antibody molecules.

Exosite 1 (also known as ‘ anion binding exosite 1’ and the ‘fibrinogenrecognition exosite’) is a well-characterized secondary binding site onthe thrombin molecule (see for example James A. Huntington, 2008,Structural Insights into the Life History of Thrombin, in RECENTADVANCES IN THROMBOSIS AND HEMOSTASIS 2008, editors; K. Tanaka and E. W.Davie, Springer Japan KK, Tokyo, pp. 80-106). Exosite 1 is formed inmature thrombin but is not formed in prothrombin (see for exampleAnderson et al (2000) JBC 2775 16428-16434).

Exosite 1 is involved in recognizing thrombin substrates, such asfibrinogen, but is remote from the catalytic active site. Variousthrombin binding factors bind to exosite 1, including the anticoagulantdodecapeptide hirugen (Naski et al 1990 JBC 265 13484-13489), factor V,factor VIII, thrombomodulin (cofactor for protein C and TAFIactivation), fibrinogen, PAR1 and fibrin (the co-factor for factor XIIIactivation).

An anti-exosite 1 antibody may bind to exosite 1 of mature humanthrombin. The sequence of human preprothrombin is set out in SEQ IDNO: 1. Human prothrombin has the sequence of residues 44 to 622 of SEQID NO: 1. Mature human thrombin has the sequence of residues 314-363(light chain) and residues 364 to 622 (heavy chain).

In some embodiments, an anti-exosite 1 antibody may also bind to exosite1 of mature thrombin from other species. Thrombin sequences from otherspecies are known in the art and available on public databases such asGenbank. The corresponding residues in thrombin sequences from otherspecies may be easily identified using sequence alignment tools.

The numbering scheme for thrombin residues set out herein isconventional in the art and is based on the chymotrypsin template (BodeW et al EMBO J. 1989 November; 8(11):3467-75). Thrombin has insertionloops relative to chymotrypsin that are lettered sequentially usinglower case letters.

Exosite 1 of mature human thrombin is underlined in SEQ ID NO: 1 and mayinclude the following residues: M32, F34, R35, K36, S36a, P37, Q38, E39,L40, L65, R67, S72, R73, T74, R75, Y76, R77a, N78, E80, K81, 182, S83,M84, K109, K110, K149e, G150, Q151, S153 and V154. In some embodiments,other thrombin residues which are located close to (i.e. within 0.5 nmor within 1 nm) of any one of these residues may also be considered tobe part of exosite 1.

An anti-exosite 1 antibody may bind to an epitope which comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morethan 20 residues of exosite 1. Preferably, an anti-exosite 1 antibodybinds to an epitope which consists entirely of exosite 1 residues.

For example, an anti-exosite 1 antibody may bind to an epitope whichcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16residues selected from the group consisting of M32, F34, S36a, P37, Q38,E39, L40, L65, R67, R73, T74, R75, Y76, R77a, 182 and Q151 of humanthrombin or the equivalent residues in thrombin from another species. Insome preferred embodiments, the epitope may comprise the thrombinresidues Q38, R73, T74, Y76 and R77a and optionally one or moreadditional residues.

Anti-exosite 1 antibody molecules as described herein are specific forthrombin exosite 1 and bind to this epitope with high affinity relativeto other epitopes, for example epitopes from mammalian proteins otherthan mature thrombin. For example, an anti-exosite 1 antibody moleculemay display a binding affinity for thrombin exosite 1 which is at least500 fold, at least 1000 fold or at least 2000 fold greater than otherepitopes.

Preferably, an antibody molecule as described herein which is specificfor exosite 1 may bind to mature thrombin but display no binding orsubstantially no binding to prothrombin.

Without being bound by any theory, anti-exosite 1 antibodies may beunable to access thrombin within the core of a haemostatic clot, and aretherefore unable to affect haemostasis by interrupting normal thrombinfunction at sites of vascular injury. However, because the anti-exosite1 antibodies still bind to thrombin on the surface of the clot and inthe outer shell of the clot, thrombosis is prevented, i.e.non-haemostatic clot extension is prevented.

An anti-exosite 1 antibody molecule may have a dissociation constant forexosite 1 of less than 50 nM, less than 40 nM, less than 30 nM, lessthan 20 nM, less than 10 nM, or less than 1 nM. For example, an antibodymolecule may have an affinity for exosite 1 of 0.1 to 50 nM, e.g. 0.5 to10 nM. A suitable anti-exosite 1 antibody molecule may, for example,have an affinity for thrombin exosite 1 of about 1 nM.

Binding kinetics and affinity (expressed as the equilibrium dissociationconstant, K_(d)) of the anti-exosite 1 antibody molecules may bedetermined using standard techniques, such as surface plasmon resonancee.g. using BIAcore analysis.

An anti-exosite 1 antibody molecule as described herein may be animmunoglobulin or fragment thereof, and may be natural or partly orwholly synthetically produced, for example a recombinant molecule.

Anti-exosite 1 antibody molecules may include any polypeptide or proteincomprising an antibody antigen-binding site, including Fab, Fab₂, Fab₃,diabodies, triabodies, tetrabodies, minibodies and single-domainantibodies, including nanobodies, as well as whole antibodies of anyisotype or sub-class. Antibody molecules and methods for theirconstruction and use are described, in for example Holliger & Hudson,Nature Biotechnology 23(9):1126-1136 (2005).

In some preferred embodiments, the anti-exosite 1 antibody molecule maybe a whole antibody. For example, the anti-exosite 1 antibody moleculemay be an IgG, IgA, IgE or IgM or any of the isotype sub-classes,particularly IgG1 and IgG4. The anti-exosite 1 antibody molecules may bemonoclonal antibodies. In other preferred embodiments, the anti-exosite1 antibody molecule may be an antibody fragment.

Anti-exosite 1 antibody molecules may be chimeric, humanized or humanantibodies.

Anti-exosite 1 antibody molecules as described herein may be isolated,in the sense of being free from contaminants, such as antibodies able tobind other polypeptides and/or serum components. Monoclonal antibodiesare preferred for some purposes, though polyclonal antibodies may alsobe employed.

Anti-exosite 1 antibody molecules may be obtained using techniques whichare standard in the art. Methods of producing antibodies includeimmunizing a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep ormonkey) with the protein or a fragment thereof. Antibodies may beobtained from immunized animals using any of a variety of techniquesknown in the art, and screened, preferably using binding of antibody toantigen of interest. For instance, Western blotting techniques orimmunoprecipitation may be used (Armitage et al., 1992, Nature 357:80-82). Isolation of antibodies and/or antibody-producing cells from ananimal may be accompanied by a step of sacrificing the animal.

As an alternative or supplement to immunizing a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047. The library may be naive, that is constructedfrom sequences obtained from an organism which has not been immunizedwith any of the proteins (or fragments), or may be one constructed usingsequences obtained from an organism which has been exposed to theantigen of interest.

Other anti-exosite 1 antibody molecules may be identified by screeningpatient serum for antibodies which bind to exosite 1.

In some embodiments, anti-thrombin antibody molecules may be produced byany convenient means, for example a method described above, and thenscreened for differential binding to mature thrombin relative tothrombin with an exosite 1 mutation, gamma thrombin (exosite 1 defectivedue to autolysis at R75 and R77a) or prothrombin. Suitable screeningmethods are well-known in the art.

An antibody which displays increased binding to mature thrombin,relative to non-thrombin proteins, thrombin with an exosite 1 mutation,gamma-thrombin or prothrombin, for example an antibody which binds tomature thrombin but does not bind to thrombin with an exosite 1mutation, gamma thrombin or prothrombin, may be identified as ananti-exosite 1 antibody molecule.

After production and/or isolation, the biological activity of ananti-exosite 1 antibody molecule may be tested. For example, the abilityof the antibody molecule to inhibit thrombin substrate, cofactor orinhibitor binding and/or cleavage by thrombin may be determined and/orthe ability of the antibody molecule to inhibit thrombosis withoutpromoting bleeding may be determined.

Suitable antibody molecules may be tested for activity using afibrinogen clotting or thrombin time assay. Suitable assays arewell-known in the art.

The effect of an antibody molecule on coagulation and bleeding may bedetermined using standard techniques. For example, the effect of anantibody molecule on thrombosis may be determined in an animal model,such as a mouse model with ferric chloride induced clots in bloodvessels. Effects on haemostasis may also be determined in an animalmodel, for example, by measuring tail bleed of a mouse.

Antibody molecules normally comprise an antigen binding domaincomprising an immunoglobulin heavy chain variable domain (VH) and animmunoglobulin light chain variable domain (VL), although antigenbinding domains comprising only a heavy chain variable domain (VH) arealso possible (e.g. camelid or shark antibodies).

Each of the VH and VL domains typically comprise three complementaritydetermining regions (CDRs) responsible for antigen binding, interspersedby framework regions.

In some embodiments, binding to exosite 1 may occur wholly orsubstantially through the VHCDR3 of the anti-exosite 1 antibodymolecule.

For example, an anti-exosite 1 antibody molecule may comprise a VHdomain comprising a HCDR3 having the amino acid sequence of SEQ ID NO: 5or the sequence of SEQ ID NO: 5 with 1 or more, for example 2, 3, 4 or 5or more amino acid substitutions, deletions or insertions. Thesubstitutions may be conservative substitutions. In some embodiments,the HCDR3 may comprise the amino acid residues at positions 4 to 9 ofSEQ ID NO: 5 (SEFEPF), or more preferably the amino acid residues atpositions 2, and 4 to 10 of SEQ ID NO: 5 (D and SEFEPFS) withsubstitutions, deletions or insertions at one or more other positions inSEQ ID NO:5. The HCDR3 may be the only region of the antibody moleculethat interacts with a thrombin exosite 1 epitope or substantially theonly region. The HCDR3 may therefore determine the specificity and/oraffinity of the antibody molecule for the exosite 1 region of thrombin.

The VH domain of an anti-exosite 1 antibody molecule may additionallycomprise an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or thesequence of SEQ ID NO: 4 with 1 or more, for example 2, 3, 4 or 5 ormore amino acid substitutions, deletions or insertions. In someembodiments, the HCDR2 may comprise the amino acid residues at positions3 to 7 of SEQ ID NO: 4 (DPQDG) or the amino acid residues at positions 2and 4 to 7 of SEQ ID NO: 4 (L and PQDG) of SEQ ID NO: 4, withsubstitutions, deletions or insertions at one or more other positions inSEQ ID NO: 4.

The VH domain of an anti-exosite 1 antibody molecule may furthercomprise an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or thesequence of SEQ ID NO: 3 with 1 or more, for example 2, 3, 4 or 5 ormore amino acid substitutions, deletions or insertions. In someembodiments, the HCDR1 may comprise amino acid residue T at position 5of SEQ ID NO: 3 with substitutions, deletions or insertions at one ormore other positions in SEQ ID NO: 3.

In some embodiments, an antibody molecule may comprise a VH domaincomprising a HCDR1, a HCDR2 and a HCDR3 having the sequences of SEQ IDNOs 3, 4 and 5 respectively. For example, an antibody molecule maycomprise a VH domain having the sequence of SEQ ID NO: 2 or the sequenceof SEQ ID NO: 2 with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10or more amino acid substitutions, deletions or insertions in SEQ ID NO:2.

The anti-exosite 1 antibody molecule may further comprise a VL domain,for example a VL domain comprising LCDR1, LCDR2 and LCDR3 having thesequences of SEQ ID NOs 7, 8 and 9 respectively, or the sequences of SEQID NOs 7, 8 and 9 respectively with, independently, 1 or more, forexample 2, 3, 4 or 5 or more amino acid substitutions, deletions orinsertions. The substitutions may be conservative substitutions. Forexample, an antibody molecule may comprise a VL domain having thesequence of SEQ ID NO: 6 or the sequence of SEQ ID NO: 6 with 1 or more,for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions,deletions or insertions in SEQ ID NO: 6.

In some embodiments, the VL domain may comprise Tyr49.

The anti-exosite 1 antibody molecule may for example comprise one ormore amino acid substitutions, deletions or insertions which improve oneor more properties of the antibody, for example affinity, functionalhalf-life, on and off rates.

The techniques that are required in order to introduce substitutions,deletions or insertions within amino acid sequences of CDRs, antibody VHor VL domains and antibodies are generally available in the art. Variantsequences may be made, with substitutions, deletions or insertions thatmay or may not be predicted to have a minimal or beneficial effect onactivity, and tested for ability to bind exosite 1 of thrombin and/orfor any other desired property.

In some embodiments, anti-exosite 1 antibody molecule may comprise a VHdomain comprising a HCDR1, a HCDR2 and a HCDR3 having the sequences ofSEQ ID NOs 3, 4, and 5, respectively, and a VL domain comprising aLCDR1, a LCDR2 and a LCDR3 having the sequences of SEQ ID NOs 7, 8 and9, respectively.

For example, the VH and VL domains may have the amino acid sequences ofSEQ ID NO: 2 and SEQ ID NO: 6 respectively; or may have the amino acidsequences of SEQ ID NO: 2 and SEQ ID NO: 6 comprising, independently 1or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsubstitutions, deletions or insertions. The substitutions may beconservative substitutions.

In some embodiments, an antibody may comprise one or more substitutions,deletions or insertions which remove a glycosylation site. For example,a glycosylation site in VL domain of SEQ ID NO: 6 may be mutated out byintroducing a substitution at either N28 or S30.

The anti-exosite 1 antibody molecule may be in any format, as describedabove, In some preferred embodiments, the anti-exosite 1 antibodymolecule may be a whole antibody, for example an IgG, such as IgG1 orIgG4, IgA, IgE or IgM.

An anti-exosite 1 antibody molecule of the invention may be one whichcompetes for binding to exosite 1 with an antibody molecule describedabove, for example an antibody molecule which

-   -   (i) binds thrombin exosite 1 and    -   (ii) comprises a VH domain of SEQ ID NO: 2 and/or VL domain of        SEQ ID NO: 6; an HCDR3 of SEQ ID NO: 5; an HCDR1, HCDR2, LCDR1,        LCDR2, or LCDR3 of SEQ ID NOS: 3, 4, 7, 8 or 9 respectively; a        VH domain comprising HCDR1, HCDR2 and HCDR3 sequences of SEQ ID        NOS: 3, 4 and 5 respectively; and/or a VH domain comprising        HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4 and 5 and a        VL domain comprising LCDR1, LDR2 and LCDR3 sequences of SEQ ID        NOS: 7, 8 and 9 respectively.

Competition between antibody molecules may be assayed easily in vitro,for example using ELISA and/or by tagging a specific reporter moleculeto one antibody molecule which can be detected in the presence of one ormore other untagged antibody molecules, to enable identification ofantibody molecules which bind the same epitope or an overlappingepitope. Such methods are readily known to one of ordinary skill in theart. Thus, a further aspect of the present invention provides anantibody molecule comprising a antibody antigen-binding site thatcompetes with an antibody molecule, for example an antibody moleculecomprising a VH and/or VL domain, CDR e.g. HCDR3 or set of CDRs of theparent antibody described above for binding to exosite 1 of thrombin. Asuitable antibody molecule may comprise an antibody antigen-binding sitewhich competes with an antibody antigen-binding site for binding toexosite 1 wherein the antibody antigen-binding site is composed of a VHdomain and a VL domain, and wherein the VH and VL domains compriseHCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4, and 5 and LCDR1,LDR2 and LCDR3 sequences of SEQ ID NOS: 7, 8, and 9 respectively, forexample the VH and VL domains of SEQ ID NOS: 2 and 6.

An anti-exosite 1 antibody molecule as described herein may inhibit thebinding of thrombin-binding factors, including factors which bind toexosite 1. For example, an antibody molecule may competitively ornon-competitively inhibit the binding of one or more of fV, fVIII,thrombomodulin, fibrinogen or fibrin, PAR1 and/or hirugen and hirudinanalogues to thrombin.

An anti-exosite 1 antibody molecule as described herein may inhibit oneor more activities of thrombin. For example, an anti-exosite 1 antibodymolecule may inhibit the hydrolytic cleavage of one or more thrombinsubstrates, such as fibrinogen, platelet receptor PAR-1 and coagulationfactor FVIII. For example, binding of the antibody molecule to thrombinmay result in an at least 5-fold, at least 10-fold, or at least 15-folddecrease in the hydrolysis of fibrinogen, PAR-1, coagulation factorFVIII and/or another thrombin substrates, such as factor V, factor XIIIin the presence of fibrin, and protein C and/or TAFI in the presence ofthrombomodulin. In some embodiments, binding of thrombin by theanti-exosite 1 antibody molecule may result in no detectable cleavage ofthe thrombin substrate by thrombin.

Techniques for measuring thrombin activity, for example by measuring thehydrolysis of thrombin substrates in vitro are standard in the art andare described herein.

Anti-exosite 1 antibody molecules may be further modified by chemicalmodification, for example by PEGylation, or by incorporation in aliposome, to improve their pharmaceutical properties, for example byincreasing in vivo half-life.

The effect of an anti-exosite 1 antibody molecule on coagulation andbleeding may be determined using standard techniques. For example, theeffect of an antibody on a thrombosis model may be determined. Suitablemodels include ferric chloride clot induction in blood vessels in amurine model, followed by a tail bleed to test normal haemostasis. Othersuitable thrombosis models are well known in the art (see for exampleWestrick et al ATVB (2007) 27:2079-2093)

Anti-exosite 1 antibody molecules may be comprised in pharmaceuticalcompositions with a pharmaceutically acceptable excipient.

A pharmaceutically acceptable excipient may be a compound or acombination of compounds entering into a pharmaceutical compositionwhich does not provoke secondary reactions and which allows, forexample, facilitation of the administration of the anti-exosite 1antibody molecule, an increase in its lifespan and/or in its efficacy inthe body or an increase in its solubility in solution. Thesepharmaceutically acceptable vehicles are well known and will be adaptedby the person skilled in the art as a function of the mode ofadministration of the anti-exosite 1 antibody molecule.

In some embodiments, anti-exosite 1 antibody molecules may be providedin a lyophilized form for reconstitution prior to administration. Forexample, lyophilized antibody molecules may be re-constituted in sterilewater and mixed with saline prior to administration to an individual.

Anti-exosite 1 antibody molecules will usually be administered in theform of a pharmaceutical composition, which may comprise at least onecomponent in addition to the antibody molecule. Thus pharmaceuticalcompositions may comprise, in addition to the anti-exosite 1 antibodymolecule, a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the anti-exosite 1 antibody molecule. The precise nature ofthe carrier or other material will depend on the route ofadministration, which may be by bolus, infusion, injection or any othersuitable route, as discussed below.

For parenteral, for example sub-cutaneous or intra-venousadministration, e.g. by injection, the pharmaceutical compositioncomprising the anti-exosite 1 antibody molecule may be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles, such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilizers,buffers, antioxidants and/or other additives may be employed as requiredincluding buffers such as phosphate, citrate and other organic acids;antioxidants, such as ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3′-pentanol; and m-cresol); low molecularweight polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagines, histidine,arginine, or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose or dextrins; chelating agents,such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions, such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG).

A pharmaceutical composition comprising an anti-exosite 1 antibodymolecule may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

An anti-exosite 1 antibody molecule as described herein may be used in amethod of treatment of the human or animal body, including prophylacticor preventative treatment (e.g. treatment before the onset of acondition in an individual to reduce the risk of the condition occurringin the individual; delay its onset; or reduce its severity after onset).The method of treatment may comprise administering an anti-exosite 1antibody molecule to an individual in need thereof.

Administration is normally in a “therapeutically effective amount”, thisbeing sufficient to show benefit to a patient. Such benefit may be atleast amelioration of at least one symptom. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated, the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the composition, themethod of administration, the scheduling of administration and otherfactors known to medical practitioners. Prescription of treatment, e.g.decisions on dosage etc., is within the responsibility of generalpractitioners and other medical doctors and may depend on the severityof the symptoms and/or progression of a disease being treated.Appropriate doses of antibody molecules are well known in the art(Ledermann J. A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922). Specific dosages may be indicated herein or in the Physician'sDesk Reference (2003) as appropriate for the type of medicament beingadministered may be used. A therapeutically effective amount or suitabledose of an antibody molecule may be determined by comparing its in vitroactivity and in vivo activity in an animal model. Methods forextrapolation of effective dosages in mice and other test animals tohumans are known. The precise dose will depend upon a number of factors,including whether the antibody is for prevention or for treatment, thesize and location of the area to be treated, the precise nature of theantibody (e.g. whole antibody, fragment) and the nature of anydetectable label or other molecule attached to the antibody.

A typical antibody dose will be in the range 100 μg to 1 g for systemicapplications, and 1 μg to 1 mg for topical applications. An initialhigher loading dose, followed by one or more lower doses, may beadministered. Typically, the antibody will be a whole antibody, e.g. theIgG1 or IgG4 isotype. This is a dose for a single treatment of an adultpatient, which may be proportionally adjusted for children and infants,and also adjusted for other antibody formats in proportion to molecularweight. Treatments may be repeated at daily, twice-weekly, weekly ormonthly intervals, at the discretion of the physician. The treatmentschedule for an individual may be dependent on the pharmocokinetic andpharmacodynamic properties of the antibody composition, the route ofadministration and the nature of the condition being treated.

Treatment may be periodic, and the period between administrations may beabout two weeks or more, e.g. about three weeks or more, about fourweeks or more, about once a month or more, about five weeks or more, orabout six weeks or more. For example, treatment may be every two to fourweeks or every four to eight weeks. Treatment may be given before,and/or after surgery, and/or may be administered or applied directly atthe anatomical site of surgical treatment or invasive procedure.Suitable formulations and routes of administration are described above.

In some embodiments, anti-exosite 1 antibody molecules as describedherein may be administered as sub-cutaneous injections. Sub-cutaneousinjections may be administered using an auto-injector, for example forlong term prophylaxis/treatment.

In some preferred embodiments, the therapeutic effect of theanti-exosite 1 antibody molecule may persist for several half-lives,depending on the dose. For example, the therapeutic effect of a singledose of anti-exosite 1 antibody molecule may persist in an individualfor 1 month or more, 2 months or more, 3 months or more, 4 months ormore, 5 months or more, or 6 months or more.

Anti-exosite 1 antibody molecules described herein inhibit thrombin andmay be useful in the treatment of thrombin-mediated conditions.

Haemostasis is the normal coagulation response i.e. the prevention ofbleeding or haemorrhage, for example from a damaged blood vessel.Haemostasis arrests bleeding and haemorrhage from blood vessels in thebody.

Anti-exosite 1 antibody molecules may have no effect or substantially noeffect on haemostasis i.e. they do not promote bleeding or haemorrhage.

Aspects of the invention provide; an anti-exosite 1 antibody molecule asdescribed herein for use in a method of treatment of the human or animalbody; an anti-exosite 1 antibody molecule as described herein for use ina method of treatment of a thrombin-mediated disorder; the use of ananti-exosite 1 antibody molecule as described herein in the manufactureof a medicament for the treatment of a thrombin-mediated condition; anda method of treatment of a thrombin-mediated condition comprisingadministering an anti-exosite 1 antibody molecule as described herein toan individual in need thereof.

Inhibition of thrombin by anti-exosite 1 antibodies as described hereinmay be of clinical benefit in the treatment of any thrombin-mediatedcondition. A thrombin-mediated condition may include disordersassociated with the formation or activity of thrombin.

Thrombin plays a key role in haemostasis, coagulation and thrombosis.Thrombin-mediated conditions include thrombotic conditions, such asthrombosis and embolism.

Thrombosis is coagulation which is in excess of what is required forhaemostasis (i.e. excessive coagulation), or which is not required forhaemostasis (i.e. extra-haemostatic or non-haemostatic coagulation).

Thrombosis is blood clotting within the blood vessel lumen. It ischaracterized by the formation of a clot (thrombus) that is in excess ofrequirement or not required for haemostasis. The clot may impede bloodflow through the blood vessel leading to medical complications. A clotmay break away from its site of formation, leading to embolism elsewherein the circulatory system. In the arterial system, thrombosis istypically the result of atherosclerotic plaque rupture.

In some embodiments, thrombosis may occur after an initial physiologicalhaemostatic response, for example damage to endothelial cells in a bloodvessel. In other embodiments, thrombosis may occur in the absence of anyphysiological haemostatic response.

Thrombosis may occur in individuals with an intrinsic tendency tothrombosis (i.e. thrombophilia) or in ‘normal’ individuals with nointrinsic tendency to thrombosis, for example in response to anextrinsic stimulus.

Thrombosis and embolism may occur in any vein, artery or other bloodvessel within the circulatory system and may include microvascularthrombosis.

Thrombosis and embolism may be associated with surgery (either duringsurgery or afterwards) or the insertion of foreign objects, such ascoronary stents, into a patient.

For example, anti-exosite 1 antibodies as described herein may be usefulin the surgical and other procedures in which blood is exposed toartificial surfaces, such as open heart surgery and dialysis.

Thrombotic conditions may include thrombophilia, thrombotic stroke andcoronary artery occlusion.

Patients suitable for treatment as described herein include patientswith conditions in which thrombosis is a symptom or a side-effect oftreatment or which confer an increased risk of thrombosis or patientswho are predisposed to or at increased risk of thrombosis, relative tothe general population. For example, an anti-exosite 1 antibody moleculeas described herein may also be useful in the treatment or prevention ofvenous thrombosis in cancer patients, and in the treatment or preventionof hospital-acquired thrombosis, which is responsible for 50% of casesof venous thromboembolism.

Anti-exosite 1 antibody molecules as described herein may exert atherapeutic or other beneficial effect on thrombin-mediated conditions,such as thrombotic conditions, without substantially inhibiting orimpeding haemostasis. For example, the risk of haemorrhage in patientstreated with anti-exosite 1 antibody molecules may not be increased orsubstantially increased relative to untreated individuals.

Individuals treated with conventional anticoagulants, such as naturaland synthetic heparins, warfarin, direct serine protease inhibitors(e.g. argatroban, dabigatran, apixaban, and rivaroxaban), hirudin andits derivatives (e.g. lepirudin and bivalirudin), and anti-plateletdrugs (e.g. clopidogrel, ticlopidine and abciximab) cause bleeding. Therisk of bleeding in patients treated with anti-exosite 1 antibodymolecules as described herein may be reduced relative to individualstreated with conventional anticoagulants.

Thrombin-mediated conditions include non-thrombotic conditionsassociated with thrombin activity, including inflammation, infection,tumor growth and metastasis, organ rejection and dementia (vascular andnon-vascular, e.g. Alzheimer's disease) (Licari et al J Vet Emerg CritCare (San Antonio). 2009 February; 19(1):11-22; Tsopanoglou et al EurCytokine Netw. 2009 Dec. 1; 20(4):171-9).

Anti-exosite 1 antibody molecules as described herein may also be usefulin in vitro testing, for example in the analysis and characterization ofcoagulation, for example in a sample obtained from a patient.

Anti-exosite 1 antibody molecules may be useful in the measurement ofthrombin generation. Assays of thrombin generation are technicallyproblematic because the conversion of fibrinogen to fibrin causesturbidity, which precludes the use of a simple chromogenic end-point.

The addition of an anti-exosite 1 antibody molecule as described hereinto a sample of blood prevents or inhibits fibrin formation and henceturbidity and permits thrombin generation to be measured using achromogenic substrate, without the need for a defibrination step.

For example, a method of measuring thrombin generation may comprisecontacting a blood sample with a chromogenic thrombin substrate in thepresence of an anti-exosite 1 antibody molecule as described herein andmeasuring the chromogenic signal from the substrate; wherein thechromogenic signal is indicative of thrombin generation in the sample.

The chromogenic signal may be measured directly without defibrination ofthe sample.

Suitable substrates are well known in the art and include 52238(H-D-Phe-Pip-Arg-pNa), β-Ala-Gly-Arg-p-nitroanilide diacetate (Prasa, D.et al. (1997) Thromb. Haemost. 78, 1215; Sigma Aldrich Inc) andTos-Gly-Pro-Arg-pNa.AcOH (Biophen CS-01(81); Aniara Inc OH USA).

Anti-exosite 1 antibody molecules may also be useful in inhibiting orpreventing the coagulation of blood as described above in extracorporealcirculations, such as haemodialysis and extracorporeal membraneoxygenation.

For example, a method of inhibiting or preventing blood coagulation invitro or ex vivo may comprise introducing an anti-exosite 1 antibodymolecule as described herein to a blood sample. The blood sample may beintroduced into an extracorporeal circulation system before,simultaneous with or after the introduction of the anti-exosite 1antibody and optionally subjected to treatment such as haemodialysis oroxygenation. In some embodiments, the treated blood may be subsequentlyadministered to an individual. Other embodiments provide an anti-exosite1 antibody molecule as described herein for use in a method ofinhibiting or preventing blood coagulation in a blood sample ex vivo andthe use of an anti-exosite 1 antibody molecule as described herein inthe manufacture of a medicament for use in a method of inhibiting orpreventing blood coagulation in a blood sample ex vivo.

Other aspects of the invention relate to the production of antibodymolecules which bind to the exosite 1 epitope of thrombin and may beuseful, for example in the treatment of pathological blood coagulationor thrombosis.

A method for producing an antibody antigen-binding domain for theexosite 1 epitope of thrombin, may comprise:

-   -   providing, by way of addition, deletion, substitution or        insertion of one or more amino acids in the amino acid sequence        of a parent VH domain comprising HCDR1, HCDR2 and HCDR3, wherein        HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID        NOS: 3, 4 and 5 respectively, a VH domain which is an amino acid        sequence variant of the parent VH domain, and;    -   optionally combining the VH domain thus provided with one or        more VL domains to provide one or more VH/VL combinations; and    -   testing said VH domain which is an amino acid sequence variant        of the parent VH domain or the VH/VL combination or combinations        to identify an antibody antigen binding domain for the exosite 1        epitope of thrombin.

A VH domain which is an amino acid sequence variant of the parent VHdomain may have the HCDR3 sequence of SEQ ID NO: 5 or a variant with theaddition, deletion, substitution or insertion of one, two, three or moreamino acids.

The VH domain which is an amino acid sequence variant of the parent VHdomain may have the HCDR1 and HCDR2 sequences of SEQ ID NOS: 3 and 4respectively, or variants of these sequences with the addition,deletion, substitution or insertion of one, two, three or more aminoacids.

A method for producing an antibody molecule that specifically binds tothe exosite 1 epitope of thrombin may comprise:

-   -   providing starting nucleic acid encoding a VH domain or a        starting repertoire of nucleic acids each encoding a VH domain,        wherein the VH domain or VH domains either comprise a HCDR1,        HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or        HCDR3 encoding region;    -   combining said starting nucleic acid or starting repertoire with        donor nucleic acid or donor nucleic acids encoding or produced        by mutation of the amino acid sequence of an HCDR1, HCDR2,        and/or HCDR3 having the amino acid sequences of SEQ ID NOS: 3, 4        and 5 respectively, such that said donor nucleic acid is or        donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3        region in the starting nucleic acid or starting repertoire, so        as to provide a product repertoire of nucleic acids encoding VH        domains; expressing the nucleic acids of said product repertoire        to produce product VH domains; optionally combining said product        VH domains with one or more VL domains;    -   selecting an antibody molecule that binds exosite 1 of thrombin,        which antibody molecule comprises a product VH domain and        optionally a VL domain; and    -   recovering said antibody molecule or nucleic acid encoding it.

Suitable techniques for the maturation and optimization of antibodymolecules are well-known in the art.

Antibody antigen-binding domains and antibody molecules for the exosite1 epitope of thrombin may be tested as described above. For example, theability to bind to thrombin and/or inhibit the cleavage of thrombinsubstrates may be determined.

The effect of an antibody molecule on coagulation and bleeding may bedetermined using standard techniques. For example, a mouse thrombosismodel of ferric chloride clot induction in a blood vessel, such as thefemoral vein or carotid artery, followed by a tail bleed to test normalhaemostasis, may be employed.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein byreference in their entirety.

Unless stated otherwise, antibody residues are numbered herein inaccordance with the Kabat numbering scheme.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described. Thus, the features set out above aredisclosed in all combinations and permutations.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures and tables describedbelow.

EXPERIMENTS

1. Antibody Isolation and Characterization

Coagulation screening was carried out on a blood plasma sample from apatient. The coagulation tests were performed on a patient who sufferedsubdural haematoma following head injury. The haematoma spontaneouslyresolved without intervention. There was no previous history of bleedingand in the 4 years since the patient presented, there have been nofurther bleeding episodes. The results are shown in Table 1.

The prothrombin time (PT), activated partial thromboplastin time (APTT),and thrombin time (TT) were all prolonged in the patient compared tocontrols, but reptilase time was normal.

Thrombin time was not corrected by heparinase, indicating that heparintreatment or contamination was not responsible. Fibrinogen levels werenormal in the patient, according to ELISA and Reptilase assays. TheClauss assay gave an artifactally low fibrinogen level due to thepresence of the thrombin inhibitor. The PT and APTT clotting times werefound to remain prolonged following a mixing test using a 50:50 mix withpooled plasma from normal individuals. This showed the presence of aninhibitor in the sample from the patient.

The patient's blood plasma was found to have a high titre of an IgA.This IgA molecule was found to bind to a human thrombin column (FIGS. 1Aand 1B). IgA binding lectin-agarose pulled down thrombin in the presencebut not the absence of the IgA. Prothrombin was not pulled down by thelectin-agarose in the presence of the IgA, indicating that the IgAspecifically binds to thrombin but not prothrombin (FIG. 2).

The binding site of the IgA on the thrombin molecule was theninvestigated.

A slightly higher rate of cleavage of S2238 by thrombin was measured inthe presence of the IgA, indicating that the IgA does not block theactive site of thrombin (FIG. 3).

The binding of fluorescently labelled hirugen to thrombin is inhibitedby the presence of 700 nM of the IgA, indicating that the epitope forthe antibody overlaps with the binding site of hirugen on thrombin,namely the exosite 1 of thrombin (FIG. 4).

The effect of the IgA on the hydrolysis of some of thrombin'sprocoagulant substrates was tested. The results are shown in Table 2.These results demonstrate that the IgA molecule isolated from thepatient sample inhibits multiple procoagulant activities of thrombin.

Inhibition of thrombin by antithrombin (AT) in the presence of the IgAwas only marginally affected in both the absence and presence of heparin(Table 3).

The dissociation constant (K_(d)) of the IgA for thrombin was initiallyestimated based on rate of 52238 hydrolysis to be approximately 12 nM(FIG. 5). The K_(d) for the binding of the IgA to S195A thrombin(inactivated by mutation of the catalytic serine) was determined to be 2nM using the ForteBio® Octet Red instrument (Table 4).

The purified IgA was cleaved with papain (FIG. 6), and the Fab fragmentwas isolated and combined with human PPACK-Thrombin (PPACK is a covalentactive site inhibitor). The human PPACK-Thrombin-FAB complex wascrystallized and used for structural analysis. The statistics of thestructure obtained were as follows: resolution is 1.9 Å; Rfactor=19.43%;Rfree=23.42%; one complex in the asymmetric unit; Ramachandran:favoured=97.0%, outliers=0%. The crystal structure revealed a closeassociation between the HCDR3 of the IgA Fab and the exosite 1 ofthrombin (FIG. 7).

In particular, residues M32, F34, Q38, E39, L40, L65, R67, R73, T74,R75, Y76, R77a and 182 of the exosite 1 all directly interact with theHCDR3 loop of the IgA Fab (FIG. 8).

PISA analysis of the antibody-thrombin interface showed that the totalburied surface area in the complex is 1075 Å². The contact residues inthe IgA heavy chain were (Kabat numbering): 30, 51, 52a, 53-55, 96, 98,99, 100, 100a, 100b, 100c, 100d). These are all in CDRs:

(SEQ ID NO: 3) CDRH1-GYTLTEAAIH; (SEQ ID NO: 4) CDRH2-GLDPQDGETVYAQQFKG;(SEQ ID NO: 5) CDRH3-GDFSEFEPFSMDYFHF(underlined residues contacting). CDRH3 was found to be the mostimportant, providing 85% of the buried surface area on the antibody. Thelight chain made one marginal contact with Tyr49, right before CDRL2(with Ser36a of thrombin). Some individual contributions to buriedsurface were: Glu99 54 Å², Phe100 134.8 Å²2, Glu100a 80.6 Å², Phe100c141.7 Å².

The contact residues in thrombin were found to be (chymotrypsinnumbering): 32, 34, 36a-40, 65, 67, 73-76, 77a, 82, and 151. The mostimportant individual contributors to the buried surface were: Gln38 86.4Å², Arg73 44.5 Å², Thr74 60.1 Å², Tyr76 78.4 Å², Arg77a 86.9 Å².

The patient did not display increased or abnormal bleeding orhaemorrhage, in spite of 3 g/l circulating levels of this IgA,demonstrating that the antibody inhibits thrombin without affectingnormal haemostasis.

2. The Effect of IgA on Animal Thrombosis Models

C57BL/6 mice were anaesthetized. A catheter was inserted in the carotidartery (for compound injection). FITC labelled fibrinogen (2 mg/ml) wasinjected via the carotid artery. PBS (control) or IgA was also injectedvia the carotid artery. The femoral vein was exposed and 10% FeCl₃applied (saturated blotting paper 3 mm in length) for 3 min to induceclotting.

Fluorescence microscopy images were taken along the length of injurysite at 0, 5, 10, and 20 min post FeCl₃ injury using fluorescencemicroscopy techniques.

Clots (fibrin deposits) in the femoral vein were clearly visible asbright areas (FIG. 9). The lowest dose of the antibody was observed tocause significant inhibition of clotting but as the dose increased,clotting was abolished (FIGS. 10 to 15).

The bleeding times of the mice were also measured. Bleeding times wereassessed as time to cessation of blood flow after a tail cut. Despitethe presence of a single outlier sample, the bleeding time was found tobe unaffected by treatment with anti-exosite 1 IgA (FIG. 16).

These results show that the anti-exosite 1 IgA antibody is a potentinhibitor of thrombosis but has no effect on bleeding time.

3. Tail Clip Assays

A tail clip assay was performed on wild-type male C57BL/6 mice injectedwith either 400 nM IgA (final concentration in blood, equivalent to adose of approximately 6 mg/Kg) or PBS. Blood loss was monitored over 10mins after the tail was cut at 3 mm diameter 15 minutes after theinjection. Total blood loss was found to be unaffected by treatment withanti-exosite 1 IgA (FIG. 17).

4. FeCl₃ Injury Carotid Artery Occlusion

FeCl₃ injury carotid artery occlusion studies were performed on 9 weekold WT C57BL/6 male mice. Mice were injected with 400 nM anti-Ha IgA(final concentration in blood, equivalent to a dose of approximately 6mg/Kg) or PBS 15 min prior to injury with 5% FeCl₃ for 2 min. Blood flowwas then monitored by Doppler and the time to occlusion measured. A“clot” was defined as stable occlusive thrombus where blood flow wasreduced to values typically less than 0.1 ml/min and stayed reduced. Inthe control mice, a stable clot was observed to form about 20 mins afterinjury (FIG. 18A). However, the majority of mice treated with 400 nManti-Ha IgA were unable to form stable clots and gave traces in whichthe clots were quickly resolved, repeatedly resolved or never formed.Three representative traces are shown in FIGS. 18B to 18D.

5. Anti-Exosite 1 IgG

The IgA molecule identified in the patient described above wasre-formatted as an IgG using standard techniques.

The clotting time of pooled human plasma spiked with increasing amountsof the original IgA and the new IgG was tested upon addition of humanthrombin to 20 nM (FIG. 19). Both parent IgA and the synthetic IgGincreased time to clot formation in an identical concentration-dependentmanner, implying identical affinities for thrombin. This was confirmedby measuring the binding of synthetic IgG to immobilized S195A thrombinusing a ForteBio® Octet Red instrument. Thrombin was attached to theprobe and the binding of the antibodies (at various concentrations) wasmonitored. On-rates and off-rates were determined. Both antibodies gavesimilar on-rates of approximately 3×10⁵ M⁻¹s⁻¹ and off-rates ofapproximately 5×10⁻⁴ s⁻¹, and dissociation constants (Kd) ofapproximately 2 nM. Kds of approximately 2 nM were also obtained for theIgA and the IgG by steady-state analysis (Table 4). A representativesteady state curve is shown in FIG. 20. The properties of the IgA weretherefore reproduced on an IgG framework.

Binding of prothrombin to the IgG antibody was tested using the Octetsystem by immobilizing IgG. Thrombin bound to the immobilized IgG withcomparable rates and affinities as those obtained using immobilizedthrombin (Table 4); prothrombin did not bind to the IgG. FIG. 21 is atrace of 24 nM thrombin binding to and dissociating from the immobilizedIgG. FIG. 22 is the same experiment using 500 nM prothrombin, and showsno evidence of binding.

TABLE 1 Coagulation Screening Results Control/ Normalized Test ResultRatio (NR) Prothrombin Time 43 sec. NR = 11-13 sec. 50:50 correction 35sec. Act. Part. 157 sec. NR = 22-23 sec. Thromboplastin Time 50:50correction 105 sec. Thrombin Time >150 sec. NR = 10-13 sec. ReptilaseTime 16 sec. Control = 15 sec. Fibrinogen Clauss 0.7 g/l NR = 1.5-4.5g/l Antigenic 5.0 g/l

TABLE 2 Effect of anti-exosite 1 IgA on thrombin hydrolysis ofprocoagulant substrates Thrombin substrate Activity Antibody EffectFibrinogen Formation of fibrin clot No detectable cleavage Plateletreceptor Activation of platelets 15-fold decrease in PAR-1 peptidehydrolysis FVIII Feedback activation of 7-fold decrease in thrombin viaXase complex hydrolysis

TABLE 3 Effect of saturating concentration of anti-exosite 1 IgA (Fab)on thrombin inhibition by antithrombin (AT) in the absence and presenceof 1 nM heparin (Hep). Rate of inhibition (M⁻¹s⁻¹) Heparin effect AT 4.8 ± 0.2 × 10³ 2.4-fold AT + Hep 11.8 ± 0.3 × 10³ AT + Fab  1.7 ± 0.1× 10³ 3.3-fold AT + Hep + Fab  5.6 ± 0.3 × 10³

TABLE 4 Binding constants of anti-exosite 1 IgA (n = 1 under thisprecise condition), IgG (n = 3) antibodies, and IgG-derived FAB to S195Athrombin (active site free, recombinant thrombin). Kd (nM)* k_(on)(M⁻¹s⁻¹) k_(off) (s⁻¹) Kd (nM)# IgA 1.8 3.3 × 10⁵ 3.7 × 10⁻⁴ 1.2 IgG 1.5± 0.3 3.3 ± 0.5 × 10⁵ 6.8 ± 1.1 × 10⁻⁴ 2.1 ± 0.3 IgG FAB ND 5.0 × 10⁵2.7 × 10⁻³ 5.3 IgG FAB⁺ 3.3 ± 0.3 4.3 × 10⁵ 2.1 × 10⁻³ 4.9 *Kddetermined from steady-state analysis of response vs. concentration. #Kdcalculated from rates. ⁺Determined using immobilized FAB.

What is claimed:
 1. A lyophilized antibody molecule comprising a VHdomain comprising a HCDR1 having the amino acid sequence of SEQ ID NO:3, a HCDR2 having the amino acid sequence of SEQ ID NO: 4, and a HCDR3having the amino acid sequence of SEQ ID NO: 5, a VL domain comprising aLCDR1 having the amino acid sequence of SEQ ID NO: 7, a LCDR2 having theamino acid sequence of SEQ ID NO: 8, and a LCDR3 having the amino acidsequence of SEQ ID NO: 9, and an IgG constant region, wherein theantibody molecule specifically binds to the exosite 1 region ofthrombin.
 2. The lyophilized antibody molecule according to claim 1,wherein the VL domain has the amino acid sequence of SEQ ID NO:
 6. 3.The lyophilized antibody molecule according to claim 1, wherein the VHdomain has the amino acid sequence of SEQ ID NO: 2.